Design of a Vitronectin-Based Recombinant Protein as a Defined Substrate for Differentiation of Human Pluripotent Stem Cells into Hepatocyte-Like Cells.
Design of a Vitronectin-Based Recombinant Protein as a Defined Substrate for Differentiation of Human Pluripotent Stem Cells into Hepatocyte-Like Cells.
复制标题
DOI：
10.1371/journal.pone.0136350
复制
发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Duncan SA
中科院分区：
文献类型：
--
作者：
Nagaoka M;Kobayashi M;Kawai C;Mallanna SK;Duncan SA
人多能干细胞（hPSC）的维持和分化通常需要在用于细胞粘附的基底上培养。常用的基质是从Engelbreth-Holm-Swarm肉瘤细胞纯化的基质胶，由细胞外基质蛋白、蛋白聚糖和生长因子的复杂混合物组成。几项研究已经成功地诱导了来自hPSC的肝细胞样细胞的分化。然而，这些研究中的大多数使用基质胶作为细胞粘附底物，这不是确定的培养条件。在试图产生支持未分化特性和分化成肝谱系细胞的基质中，我们设计了由融合至IgG Fc结构域的玻连蛋白片段组成的新型底物。hPSC通过整合素和RGD（Arg-Gly-Asp）基序之间的相互作用粘附到这些基质上，并且细胞保持其未分化的表型。使用先前建立的分化方案，hPSC在含有玻连蛋白片段的基质上有效地分化成中内胚层和肝谱系细胞。我们发现，全长玻连蛋白不支持稳定的细胞粘附在规范阶段。此外，具有最小含RGD结构域的玻连蛋白片段足以在完全限定的条件下将人诱导多能干细胞分化为肝谱系细胞，这有助于从hPSC分化的细胞的临床应用。
Maintenance and differentiation of human pluripotent stem cells (hPSCs) usually requires culture on a substrate for cell adhesion. A commonly used substratum is Matrigel purified from Engelbreth—Holm—Swarm sarcoma cells, and consists of a complex mixture of extracellular matrix proteins, proteoglycans, and growth factors. Several studies have successfully induced differentiation of hepatocyte-like cells from hPSCs. However, most of these studies have used Matrigel as a cell adhesion substrate, which is not a defined culture condition. In an attempt to generate a substratum that supports undifferentiated properties and differentiation into hepatic lineage cells, we designed novel substrates consisting of vitronectin fragments fused to the IgG Fc domain. hPSCs adhered to these substrates via interactions between integrins and the RGD (Arg-Gly-Asp) motif, and the cells maintained their undifferentiated phenotypes. Using a previously established differentiation protocol, hPSCs were efficiently differentiated into mesendodermal and hepatic lineage cells on a vitronectin fragment-containing substrate. We found that full-length vitronectin did not support stable cell adhesion during the specification stage. Furthermore, the vitronectin fragment with the minimal RGD-containing domain was sufficient for differentiation of human induced pluripotent stem cells into hepatic lineage cells under completely defined conditions that facilitate the clinical application of cells differentiated from hPSCs.